β-Adrenergic receptors (β-ARs) promote brown adipose tissue (BAT) thermogenesis by mobilizing fatty acids and inducing the expression of oxidative genes. β-AR activation increases the expression of oxidative genes by elevating cAMP, but whether lipolytic products can modulate gene expression is not known. This study examined the role that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) plays in the induction of gene expression. Activation of brown adipocytes by β-AR agonism or 8-bromo-cyclic AMP increased the expression of PGC1α, PDK4, PPARα, uncoupling protein 1 (UCP1), and neuron-derived orphan receptor-1 (NOR-1), and concurrent inhibition of HSL reduced the induction of PGC1α, PDK4, PPARα, and UCP1 but not NOR-1. Similar results were observed in the BAT of mice following pharmacological or genetic inhibition of HSL and in brown adipocytes with stable knockdown of ATGL. Conversely, treatments that increase endogenous fatty acids elevated the expression of oxidative genes. Pharmacological antagonism and siRNA knockdown indicate that PPARα and PPARδ modulate the induction of oxidative genes by β-AR agonism. Using a live cell fluorescent reporter assay of PPAR activation, we demonstrated that ligands for PPARα and -δ, but not PPARγ, were rapidly generated at the lipid droplet surface and could transcriptionally activate PPARα and -δ. Knockdown of ATGL reduced cAMP-mediated induction of genes involved in fatty acid oxidation and oxidative phosphorylation. Consequently, ATGL knockdown reduced maximal oxidation of fatty acids, but not pyruvate, in response to cAMP stimulation. Overall, the results indicate that lipolytic products can activate PPARα and PPARδ in brown adipocytes, thereby expanding the oxidative capacity to match enhanced fatty acid supply.

Our aging society is confronted with a dramatic increase of patients suffering from tauopathies, which include Alzheimer disease and certain frontotemporal dementias. These disorders are characterized by typical neuropathological lesions including hyperphosphorylation and subsequent aggregation of TAU protein and neuronal cell death. Currently, no mechanism-based cures are available. We generated fluorescently labeled TAU transgenic zebrafish, which rapidly recapitulated key pathological features of tauopathies, including phosphorylation and conformational changes of human TAU protein, tangle formation, neuronal and behavioral disturbances, and cell death. Due to their optical transparency and small size, zebrafish larvae are well suited for both in vivo imaging and drug development. TAU-induced neuronal cell death was imaged by time-lapse microscopy in vivo. Furthermore, we used this zebrafish model to identify compounds targeting the TAU kinase glycogen synthase kinase 3beta (GSK3beta). We identified a newly developed highly active GSK3beta inhibitor, AR-534, by rational drug design. AR-534 reduced TAU phosphorylation in TAU transgenic zebrafish. This transgenic zebrafish model may become a valuable tool for further studies of the neuropathology of dementia.

BACKGROUND

Members of major intrinsic proteins (MIPs) include water-conducting aquaporins and glycerol-transporting aquaglyceroporins. MIPs play important role in plant-water relations. The model plants Arabidopsis thaliana, rice and maize contain more than 30 MIPs and based on phylogenetic analysis they can be divided into at least four subfamilies. Populus trichocarpa is a model tree species and provides an opportunity to investigate several tree-specific traits. In this study, we have investigated Populus MIPs (PtMIPs) and compared them with their counterparts in Arabidopsis, rice and maize.

RESULTS

Fifty five full-length MIPs have been identified in Populus genome. Phylogenetic analysis reveals that Populus has a fifth uncharacterized subfamily (XIPs). Three-dimensional models of all 55 PtMIPs were constructed using homology modeling technique. Aromatic/arginine (ar/R) selectivity filters, characteristics of loops responsible for solute selectivity (loop C) and gating (loop D) and group conservation of small and weakly polar interfacial residues have been analyzed. Majority of the non-XIP PtMIPs are similar to those in Arabidopsis, rice and maize. Additional XIPs were identified from database search and 35 XIP sequences from dicots, fungi, moss and protozoa were analyzed. Ar/R selectivity filters of dicots XIPs are more hydrophobic compared to fungi and moss XIPs and hence they are likely to transport hydrophobic solutes. Loop C is longer in one of the subgroups of dicot XIPs and most probably has a significant role in solute selectivity. Loop D in dicot XIPs has higher number of basic residues. Intron loss is observed on two occasions: once between two subfamilies of eudicots and monocot and in the second instance, when dicot and moss XIPs diverged from fungi. Expression analysis of Populus MIPs indicates that Populus XIPs don't show any tissue-specific transcript abundance.

CONCLUSIONS

Due to whole genome duplication, Populus has the largest number of MIPs identified in any single species. Non-XIP MIPs are similar in all four plant species considered in this study. Small and weakly polar residues at the helix-helix interface are group conserved presumably to maintain the hourglass fold of MIP channels. Substitutions in ar/R selectivity filter, insertion/deletion in loop C, increasing basic nature of loop D and loss of introns are some of the events occurred during the evolution of dicot XIPs.

In 15-month-old db/db mice, signs of diabetic retinopathy, including blood-retinal barrier breakdown, loss of pericytes, neuro-retinal apoptosis, glial reactivation, and proliferation of blood vessels, were evident. These changes in the diabetic retina were associated with increased expression of aldose reductase (AR). To further understand the role of AR in the pathogenesis of diabetic retinopathy, we generated db/db mice with an AR null mutation (AR-/- db/db). AR deficiency led to fewer retinal blood vessels with IgG leakage, suggesting that AR may contribute to blood-retinal barrier breakdown. AR deficiency also prevented diabetes-induced reduction of platelet/endothelial cell adhesion molecule-1 expression and increased expression of vascular endothelial growth factor, which may have contributed to blood-retinal barrier breakdown. In addition, long-term diabetes-induced neuro-retinal stress and apoptosis and proliferation of blood vessels were less prominent in AR-/- db/db mice. These findings indicate that AR is responsible for the early events in the pathogenesis of diabetic retinopathy, leading to a cascade of retinal lesions, including blood-retinal barrier breakdown, loss of pericytes, neuro-retinal apoptosis, glial reactivation, and neovascularization.

The androgen receptor (AR) is expressed in many cell types and the androgen/AR signaling has been found to have important roles in modulating tumorigenesis and metastasis in several cancers including prostate, bladder, kidney, lung, breast and liver. However, whether AR has differential roles in the individual cells within these tumors that contain a variety of cell types remains unclear. Generation of AR knockout (ARKO) mouse models with deletion of AR in selective cells within tumors indeed have uncovered many unique AR roles in the individual cell types during cancer development and progression. This review will discuss the results obtained from various ARKO mice and different human cell lines with special attention to the cell type- and tissue-specific ARKO models. The understanding of various results showing the AR indeed has distinct and contrasting roles in each cell type within many hormone-related tumors (as stimulator in bladder, kidney and lung metastases vs as suppressor in prostate and liver metastases) may eventually help us to develop better therapeutic approaches by targeting the AR or its downstream signaling in individual cell types to better battle these hormone-related tumors in different stages.

Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase epsilon (Dpb11-Polepsilon complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polepsilon associated with these fragments. Association of Dpb11 and Polepsilon with these fragments was mutually dependent, suggesting that the Dpb11-Polepsilon complex associates with the ARS. Moreover, Dpb11 was required for the association of Polalpha-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polepsilon complex with the ARS fragments is required for the association of the Polalpha-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polepsilon in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases alpha and epsilon and the ARS.

Androgen receptor (AR) belongs to the steroid hormone nuclear receptor superfamily. It functions as an androgen-dependent transcriptional factor that regulates genes for cell proliferation and differentiation. Caveolin is a principal component of caveolae membranes serving as a scaffold protein of many signal transduction pathways. Recent results correlate caveolin-1 expression with androgen sensitivity in murine prostate cancer. Furthermore, immunohistochemical staining of patient specimens suggests that caveolin expression may be an independent predictor of progression of prostate cancer. In this study, we investigate the potential interactions between AR signaling and caveolin-1 and demonstrate that overexpression of caveolin-1 potentiates ligand-dependent AR activation. Conversely, down-regulation of caveolin-1 expression by a caveolin-1 antisense expression construct can down-regulate ligand-dependent AR activation. Association between these two molecules is also demonstrated by co-localization of AR with caveolin-rich, low-density membrane fractions isolated by an equilibrium sucrose gradient centrifugation method. Co-immunoprecipitation and glutathione S-transferase fusion protein pull-down experiments demonstrate that interaction between AR and caveolin-1 is an androgen-dependent process, offering further evidence for a physiological role of this interaction. Using a mammalian two-hybrid assay system, we determine that the NH(2) terminus region of caveolin-1 is responsible for the interaction with both the NH(2)-terminal domain and the ligand-binding domain of AR.

X-linked spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder characterized by lower motor neuron degeneration. SBMA is caused by polyglutamine repeat expansions in the androgen receptor (AR). To determine the basis of AR polyglutamine neurotoxicity, we introduced human AR yeast artificial chromosomes carrying either 20 or 100 CAGs into mouse embryonic stem cells. The ARAR interferes with CREB binding protein (CBP)-mediated transcription of vascular endothelial growth factor (VEGF) and observed altered CBP-AR binding and VEGF reduction in ARAR-induced death of motor neuron-like cells could be rescued by VEGF. Our results suggest that SBMA motor neuronopathy involves altered expression of VEGF, consistent with a role for VEGF as a neurotrophic/survival factor in motor neuron disease.

We have constructed a variety of vectors for use in both budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe). Four of these, pDB262, pWH4, pWH5, and pMAK262, have positive selection for the insertion of cloned DNA, making them convenient for the construction of gene banks. pDB262, pWH4 and pWH5 contain the 2 mu ARS and the LEU2 gene from S. cerevisiae and can be used for gene isolation. They can also be converted into integration vectors for use in the genetic mapping of cloned sequences. pMAK262 contains only the LEU2 gene from budding yeast and can be used to screen for ARS elements or for gene integration. We also describe two other integration vectors, pDAM3 and pDAM6, which have a variety of restriction sites suitable for subcloning.

We characterized a number of widely used yeast-Escherichia coli shuttle vectors in the fission yeast Schizosaccharomyces pombe. The 2 micron vectors pDB248 and YEp13 showed high frequency of transformation, intermediate mitotic and low meiotic stability, and a low copy number in S. pombe, analogous to their behavior in [cir0] strains of Saccharomyces cerevisiae. The S. cerevisiae integration vectors pLEU2 and pURA3 transformed S. pombe at very low frequencies but, surprisingly, in a nonintegrative fashion. Instead, they replicated autonomously, and they showed very high copy numbers (up to 150 copies per plasmid-containing cell). This could reflect a lack of sequence specificity for replication of plasmid DNA in S. pombe. pFL20, an S. pombe ars vector, and a series of plasmids derived from it were studied to analyze the unusually high stability of this plasmid. Mitotic stability and partitioning of the plasmids was measured by pedigree analysis of transformed S. pombe cells. An S. pombe DNA fragment (stb) was identified that stabilizes pFL20 by improvement of plasmid partitioning in mitosis and meiosis.

Functional androgen receptor (AR) signaling is necessary for the development of prostate cancer. The therapeutic effect of androgen deprivation therapy for prostate cancer was described over 60 years ago and this treatment remains the mainstay of systemic therapy despite its transient response duration. It has become clear that AR expression and signaling remains intact as the disease evolves from androgen-sensitive cancer to classically (but perhaps inaccurately) termed hormone refractory prostate cancer. Through several genetic and epigenetic adaptations, prostate tumors continue to rely on AR growth signaling and they thus remain targets of 'hormonal' therapy. The development of new strategies and drugs that can abrogate AR signaling will probably result in important clinical benefits. The biology of androgen independence and the development of new approaches targeting AR signaling are reviewed herein.

An aldol-based build/couple/pair (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti-aldol reactions were performed to produce four stereoisomers of a Boc-protected γ-amino acid. In addition, both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes: nucleophilic aromatic substitution (S(N)Ar), Huisgen [3+2] cycloaddition, and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields, providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on the solid phase to yield a 14 400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition, and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.

The sympathetic nervous system modulates cardiac contractility and rate by activating beta-adrenergic receptors (beta AR) expressed on cardiac myocytes and specialized cells in the sinoatrial node and the conduction system. Recent clinical studies have suggested that beta-adrenergic receptors also play a role in cardiac remodeling that occurs in the pathogenesis of cardiomyopathy. Both beta(1) and beta(2) adrenergic receptors are expressed in human and murine hearts. We have examined the effect of beta AR activation on the spontaneous contraction rate of neonatal myocyte cultures from wild-type and beta receptor knockout (KO) mice (beta(1)AR-KO, beta(2)AR-KO and beta(1)beta(2)AR-KO mice). Stimulation of the beta(1)AR in beta(2)AR-KO myocytes produces the greatest increase in contraction rate through a signaling pathway that requires protein kinase A (PKA) activation. In contrast, stimulation of the beta(2)AR in beta(1)AR-KO myocytes results in a biphasic effect on contraction rate with an initial increase in rate that does not require PKA, followed by a decrease in rate that involves coupling to a pertussis toxin sensitive G protein. A small isoproterenol-induced decrease in contraction rate observed in beta(1)beta(2)AR-KO myocytes can be attributed to the beta(3)AR. These studies show that all three beta AR subtypes are expressed in neonatal cardiac myocytes, and the beta(1)AR and beta(2)AR couple to distinct signaling pathways.

Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible.

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference-mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer.

Because of increasing life expectancy, the contribution of age-related estrogen or androgen deficiency to obesity and type 2 diabetes will become a new therapeutic challenge. This review integrates current concepts on the mechanisms through which estrogen receptors (ERs) and androgen receptor (AR) regulate energy homeostasis in rodents and humans. In females, estrogen maintains energy homeostasis via ERα and ERβ, by suppressing energy intake and lipogenesis, enhancing energy expenditure, and ameliorating insulin secretion and sensitivity. In males, testosterone is converted to estrogen and maintains fuel homeostasis via ERs and AR, which share related functions to suppress adipose tissue accumulation and improve insulin sensitivity. We suggest that ERs and AR could be potential targets in the prevention of age-related metabolic disorders.

The recent progress in crystallography of G-protein coupled receptors opens an unprecedented venue for structure-based GPCR drug discovery. To test efficiency of the structure-based approach, we performed molecular docking and virtual ligand screening (VLS) of more than 4 million commercially available "drug-like" and ''lead-like'' compounds against the A(2A)AR 2.6 A resolution crystal structure. Out of 56 high ranking compounds tested in A(2A)AR binding assays, 23 showed affinities under 10 microM, 11 of those had sub-microM affinities and two compounds had affinities under 60 nM. The identified hits represent at least 9 different chemical scaffolds and are characterized by very high ligand efficiency (0.3-0.5 kcal/mol per heavy atom). Significant A(2A)AR antagonist activities were confirmed for 10 out of 13 ligands tested in functional assays. High success rate, novelty, and diversity of the chemical scaffolds and strong ligand efficiency of the A(2A)AR antagonists identified in this study suggest practical applicability of receptor-based VLS in GPCR drug discovery.

BACKGROUND

In the United States, Puerto Ricans and Mexicans have the highest and lowest asthma prevalence, morbidity, and mortality, respectively. Ethnic-specific differences in the response to drug treatment may contribute to differences in disease outcomes. Genetic variants at the beta(2)-adrenergic receptor (beta(2)AR) may modify asthma severity and albuterol responsiveness. We tested the association of beta(2)AR genotypes with asthma severity and bronchodilator response to albuterol in Puerto Ricans and Mexicans with asthma.

METHODS

We used both family-based and cross-sectional tests of association with 8 beta(2)AR single nucleotide polymorphisms in 684 Puerto Rican and Mexican families. Regression analyses were used to determine the interaction between genotype, asthma severity, and bronchodilator drug responsiveness.

RESULTS

Among Puerto Ricans with asthma, the arginine (Arg) 16 allele was associated with greater bronchodilator response using both family-based and cross-sectional tests (p = 0.00001-0.01). We found a strong interaction of baseline FEV(1) with the Arg16Glycine (Gly) polymorphism in predicting bronchodilator response. Among Puerto Ricans with asthma with baseline FEV(1) < 80% of predicted, but not in those with FEV(1)>> 80%, there was a very strong association between the Arg16 genotype and greater bronchodilator responsiveness. No association was observed between Arg16Gly genotypes and drug responsiveness among Mexicans with asthma.

CONCLUSIONS

Ethnic-specific pharmacogenetic differences exist between Arg16Gly genotypes, asthma severity, and bronchodilator response in Puerto Ricans and Mexicans with asthma. These findings underscore the need for additional research on racial/ethnic differences in asthma morbidity and drug responsiveness.

Prostatic tissue specimens derived from transurethral resections of patients with metastatic prostate cancer were analyzed for genetic alterations in the hormone-binding domain of the androgen receptor (AR) gene. Direct sequencing of the polymerase chain reaction-derived DNAs of 6 of 24 specimens revealed a codon 877 mutation (ACT->>GCT, Thr->>Ala) in the hormone-binding domain of the AR gene. This same AR mutation has been reported previously in a metastatic prostate cancer cell line, LNCaP, where this mutation confers upon the AR an altered ligand-binding specificity which is stimulated by estrogens, progestagens, and antiandrogens. It is possible that analogous to an activated/altered growth factor receptor oncogene, codon 877 mutant AR with altered ligand binding may provide a selective growth advantage in the genesis of a subset of advanced prostate cancer. Although estrogens are used infrequently, antiandrogens are used increasingly in hormonal therapy for patients with advanced prostate cancer. The stimulatory effect of these therapeutic agents on the codon 877 mutant AR further suggests that this frequently observed AR mutation may contribute to the treatment refractory disease.

We report the results of pathologic and biochemical studies in a patient with 6q-linked autosomal recessive juvenile parkinsonism (AR-JP). Neuronal loss and gliosis were restricted to the substantia nigra and the locus ceruleus. No Lewy bodies were found, but neurofibrillary tangles and argyrophilic astrocytes were seen in the cerebral cortex and brainstem nuclei. The later findings, which have not been reported previously in AR-JP, suggest the pathologic heterogeneity of 6q-linked AR-JP.

Androgens are essential for the maintenance of normal spermatogenesis in the rat. We assessed the sites, developmental pattern, and hormonal control of androgen receptors (AR) in the rat testis. Adult male rats were studied after 1) no treatment; 2) ethane dimethane sulfonate (EDS), which eradicates Leydig cells and endogenous testosterone (T); 3) EDS plus T replacement beginning at the time of EDS administration; or 4) methoxyacetic acid, which leads to the loss of specific germ cell types. Testes were also obtained from normal immature rats (aged 5, 14, 16, 21, 28, 31, 35, 38, and 45 days). After microwave antigen retrieval, immunohistochemistry was performed using a rabbit polyclonal antibody (Novocastra) raised against a peptide unique to the N-terminal region of the AR and detection with biotinylated swine antirabbit immunoglobulin G, avidin-biotin complex/alkaline phosphatase, and nitroblue tetrazolium salt (NBT)/5 bromo-4-chloro-3-indolylphosphate (BCIP) substrate. In adults, nuclear immunostaining of Sertoli cells (SC) increased progressively in intensity from stages II through VII of the spermatogenic cycle, and then declined precipitously during stage VIII to become barely detectable in stages IX-XIII. Prominent AR immunostaining was also evident in peritubular myoid cells, arterioles, and interstitial cells; staining in these cells did not vary with the stage of the cycle of the adjacent tubules. EDS caused a severe loss of AR immunostaining in all cell types. Replacement of T in EDS-treated animals resulted in a pattern of AR immunostaining comparable to that in controls, although staining intensity was reduced. Methoxyacetic acid administration did not affect the pattern of AR staining. In immature rats, peritubular myoid cell immunostaining was prominent from day 5; SC staining was detectable on day 5, increased in intensity with age, and became stage dependent between days 21-35. The following conclusions were reached. 1) Immunohistochemically detectable AR expression in SC occurs predominantly in stages II-VII of the spermatogenic cycle, with highest levels at stage VII. 2) AR immunostaining is also prominent in peritubular myoid cells, arterioles, and Leydig cells (but not in germ cells), but is unrelated to the stage of adjacent tubules. 3) Endogenous T and/or its metabolites control the expression of AR in the testis. 4) AR immunostaining is detectable by day 5 of age and becomes stage specific in SC between days 21-35.

OBJECTIVE

We previously showed that intestinal inflammation is reduced by electrical stimulation of the efferent vagus nerve, which prevents postoperative ileus in mice. We propose that this cholinergic anti-inflammatory pathway is mediated via alpha7 nicotinic acetylcholine receptors expressed on macrophages. The aim of this study was to evaluate pharmacologic activation of the cholinergic anti-inflammatory pathway in a mouse model for postoperative ileus using the alpha7 nicotinic acetylcholine receptor-agonist AR-R17779.

METHODS

Mice were pretreated with vehicle, nicotine, or AR-R17779 20 minutes before a laparotomy (L) or intestinal manipulation (IM). Twenty-four hours thereafter gastric emptying was determined using scintigraphy and intestinal muscle inflammation was quantified. Nuclear factor-kappaB transcriptional activity and cytokine production was assayed in peritoneal macrophages.

RESULTS

Twenty-four hours after surgery IM led to a delayed gastric emptying compared with L (gastric retention: L(saline) 14% +/- 4% vs IM(saline) 38% +/- 10%, P = .04). Pretreatment with AR-R17779 prevented delayed gastric emptying (IM(AR-R17779) 15% +/- 4%, P = .03). IM elicited inflammatory cell recruitment (L(saline) 50 +/- 8 vs IM(saline) 434 +/- 71 cells/mm(2), P = .001) which was reduced by AR-R17779 pretreatment (IM(AR-R17779) 231 +/- 32 cells/mm(2), P = .04). An equimolar dose of nicotine was not tolerated. Subdiaphragmal vagotomy did not affect the anti-inflammatory properties of AR-R17779. In peritoneal macrophages, both nicotinic agonists reduced nuclear factor kappaB transcriptional activity and proinflammatory cytokine production, with nicotine being more effective than AR-R17779.

CONCLUSIONS

AR-R17779 treatment potently prevents postoperative ileus, whereas toxicity limits nicotine administration to ineffective doses. Our data further imply that nicotinic inhibition of macrophage activation may involve other receptors in addition to alpha7 nicotinic acetylcholine receptor.

We created transgenic mice that overexpress WT androgen receptor (AR) exclusively in their skeletal muscle fibers. Unexpectedly, these mice display androgen-dependent muscle weakness and early death, show changes in muscle morphology and gene expression consistent with neurogenic atrophy, and exhibit a loss of motor axons. These features reproduce those seen in models of Kennedy disease, a polyglutamine expansion disorder caused by a CAG repeat expansion in the AR gene. These findings demonstrate that toxicity in skeletal muscles is sufficient to cause motoneuron disease and indicate that overexpression of the WT AR can exert toxicity comparable with the polyglutamine expanded protein. This model has two clear implications for Kennedy disease: (i) mechanisms affecting AR gene expression may cause neuromuscular symptoms similar to those of Kennedy disease and (ii) therapeutic approaches targeting skeletal muscle may provide effective treatments for this disease.